The way a protein acquires its correct three-dimensional structure was hitherto thought to be a spontaneous process. The protein folding in many cases requires the action of one or more other proteins termed molecular chaperones. This proposal concerns the process of facilitated protein folding in the eukaryotic cytosol. The investigator discovered a cytoplasmic chaperonin (analogous to those found in prokaryotes and organelles) by virtue of its ability to fold denatured actin; they find that the same chaperonin, together with two protein cofactors, also facilitates the folding of alpha- and beta-tubulin. The mechanism of facilitated folding by the cytoplasmic chaperonin and the functional role of additional cofactors are completely unknown. 1) He will purify the cofactors that are required for the release of native alpha- and beta-tubulin. Purified material will be used in experiments to determine their function via biochemical assays and sequence analysis. 2) Unexpectedly, the investigators data points to a selective interaction of the cytoplasmic chaperonin with a limited subset of cytoplasmic proteins. He will attempt to identify proteins (other than actin and tubulin) that are recognized by the chaperonin. 3) He will perform experiments to define those motifs within the actin and tubulin polypeptides that are responsible for recognition by the chaperonin, a) by defining subfragments that form (or fail to form) binary complexes with chaperonin, b) via competition experiments to define the nature and specificity of the sites required for interaction, and c) by identifying fragments of target protein that are protected from proteolysis by virtue of their interaction with chaperonin. 4) Dr. Cowan will investigate the role of ATP hydrolysis in the cytosolic chaperonin-mediated folding reaction. 5) He will determine the stoichiometry of substrate binding to the cytoplasmic chaperonin. 6) He will purify and characterize cytoplasmic factors (other than chaperonin) that form complexes with newly translated actin and tubulin, and perform experiments to define the role of these factors in the overall folding pathway.